To demonstrate their applications in biological and medical fields such as in immunoassays, magnetic separation of cells or proteins, drug or gene delivery, and magnetic resonance imaging, the template-free syntheses of water-soluble and surface functionalized magnetic nanomaterials have become essential and are challenging. Herein, we developed a facile one-pot template-free method for the preparation of amine-functionalized magnetite nanoparticles and hollow nanospheres by using FeCl3⋅6 H2O as single iron source. These magnetic nanomaterials were characterized by TEM, SEM, XRD, and FTIR technologies. Their magnetic properties were also studied by using a superconducting quantum interference device (SQUID) magnetometer at room temperature. Then the amine-functionalized magnetite nanoparticles were applied to immunoassays and magnetic resonance imaging in live mice.